Acute kidney injury (AKI) and chronic kidney disease (CKD) are critical health problems in the Veteran community. In the absence of sufficient tubule repair after injury and despite the best medical therapy, decreasing kidney function often leads to dialysis and significant morbidity and mortality. The growing gap between the increasing demand and limited supply of transplantable organs is now the chief limiting obstacle preventing extension of kidney transplantation to all patients and veterans with end-stage renal disease. Stem cell biology, and in particular induced pluripotent stem cell (iPSC) technology, provides new strategies via innovative advances in genomic and personalized medicine to decode the mechanisms of renal repair and pathways influencing tissue regeneration, to offer an opportunity for future clinical intervention to repair injured renal tubules or replace failed renal tissue. We address the current limitation in stem cel technology-to delineate and target mechanisms of stem cell maturation in three-dimensional (3D) tissue. To define these fundamental parameters we developed and characterized acellular 3D scaffolds from decellularized rodent kidneys, which allow study of growth and differentiation of stem and progenitor cells toward metanephric mesenchyme, and ultimately a mature renal lineage. Evidence from our group, as well as others, indicate that signals from 3D extracellular matrix (ECM) scaffolds induce stem cell maturation. Our findings show stem cell differentiation in a geographically-specific manner with maturation and expression of E-cadherin centered around matrix-lined tubules. Our 3D scaffolds are bioactive, consisting of natural tissue- specific ECM with structural proteins (e.g. collagens, laminins) and growth factors (e.g. VEGF, bFGF) known to mediate renal organogenesis. We will test the hypothesis that signals from 3D renal ECM tissue are a prerequisite to direct proper stem and renal progenitor cell differentiation to form nephron segments. The cellular complexity of the kidney prompts us to specifically focus on tubulogenesis within acellular matrix scaffolds and determine the requirements for regeneration and tissue formation in vitro. We will use two renal stem/progenitor populations: an early kidney-derived, adult renal progenitor cell capable of intercalating into developing nephrons, and a population of pluripotent reprogrammed human iPSCs and embryonic stem cells that differentiate to form early metanephric tissue. Our overall goal using 3D scaffolds and stem cells is to elucidate the elements of stem cell-matrix interactions that drive differentiation Aim 1 will investigate stem/progenitor cell response to modification of the renal ECM in a high-throughput system using small ECM scaffolds to rapidly screen conditions favoring differentiation. In this process we identify the requisite matrix-bound growth factors and test the role of ECM remodeling by renal stroma fibroblasts. Aim 2 will assess scalability by analyzing the requirements for stem/progenitor growth in larger-scale organ-sized scaffolds and explore tubule formation within a perfusion bioreactor. This system serves as a model for nephron development within a full-scale kidney scaffold and establishes the requirements for stem cell differentiation and scaffold repopulation within normal and diseased ECM. This investigative strategy uses innovative matrix technology to modify tissue scaffolds with fibroblasts and bioactive ligands to delineate mechanisms of differentiation in 3D. Our systematic investigation is highly significant because it will decode the critical factors involved in nephron reconstitutio, which are critical next steps in tubule repair and renal tissue regeneration.